The primary goal of our research is to understand the mechanisms involved in DNA replication in eukaryotic cells. We have chosen to study these processes using the herpesviruses as a model, since this affords the opportunity to not only gain new insights into the process of DNA replication itself, but also to learn more about the biology of an important human pathogen. We have determined the underlying biochemical defect in a mutant of herpes simplex virus that was originally characterized by lack of neurovirulence in mice. Many wild-type strains of both HSV-1 and HSV-2 are highly virulent when intracranially into mice, but a number of mutant strains of HSV with defects in virulence have been identified and have been used to attempt to gain an understanding of the genetic and molecular basis for HSV pathogenesis One such virus is R13-1, an HSV-1/HSV-2 intertypic recombinant virus in which the left-hand 18% of the genome (around 30 kb) is derived from the HG52 strain of HSV-2 and the remainder is derived from HSV-1 17syn+ strain. As far as is known, the DNA sequence of both the HSV-1 and HSV-2 segments of R13-1 are identical to their respective parental genomes. Nevertheless, studies on the phenotype of R13-1 show that it is 10,000-fold less neurovirulent in mice than either the wild type HSV-1 (strain 17+) or HSV-2 (strain HG52) parental. Moreover, R13-1 progress into the nervous system following footpad inoculation is primarily restricted at the level of spinal ganglia and viral antigen is detected in supporting cells but in few neurons. In cell culture experiments, R13-1 replicates to near wild-type levels in Vero cells, primary mouse glial cells, or Rat-2 fibroblast cells, but is restricted in primary neurons and in rat pheochromocytoma PC12 cells. The inability of R13-1 to replicate in neuronal cells can be accounted for by a specific defect in viral DNA synthesis. In neuronal cells, immediate early and early gene expression occur at wild type levels, but DNA replication and late gene expression are greatly reduced. In contrast, in non-neuronal cells both DNA replication and late gene expression occur at the same levels as seen with the wild-type parental virus. Our results have clearly shown that the defect in this mutant can be traced to the expression of an intertypic helicase primase complex. The helicase primase complex produced by this virus has two subunits derived from HSV-1 and one subunit derived from HSV-2. These two viruses are very closely related--in fact DNA sequence analysis has shown that the genes of the two viruses are approximately 95% identical. Our results suggest, however, that the 5% difference in amino acid sequence of the helicae subunit has no effect on the helicase activity but does lead to a somewhat defective interaction with the primase subunit so that primase activity is quantitatively reduced. These findings have two implications. First, they support and extend some of our previous results which showed that interactions between the subunits of the helicase primase are important for the enzymatic function of the subunits. Second, they suggest that the details of viral DNA replication in neuronal cells is different that an other types of cells, since the ten-fold decrease in primase activity has no effect on the growth of the virus in non-neuronal cells but has a profound effect on virus replication in neronal cells. It is likely that understanding why R13-1 cannot replicate DNA in neurons will lead to a better understanding of the control of virus replication during natural infections.